Conduction block (CB), a failure of action potential propagation along the nerve, causes neurological disabilities in a number of demyelinating diseases of the central and peripheral nervous systems, including Guillain-Barre syndrome, chronic inflammatory demyelinating polyneuropathy, and multiple sclerosis. The molecular basis for CB, however, is not well understood. Interestingly, patients with hereditary neuropathy with liability to pressure palsies (HNPP), an inherited condition with a deletion of one copy of chromosome 17p11.2 containing the PMP22 gene, are abnormally sensitive to mechanical force on the peripheral nerve, and develop reversible focal weakness and sensory loss which are probably due to CB. In the past 4 years, through the support of an NIH K08 grant, the PI's laboratory has studied CB using an authentic animal model of HNPP, the pmp22 heterozygous knockout mouse (pmp22). We found that CB can be mechanically induced more rapidly in the pmp22 mice than that in wild-type mice. We have identified frequent focal axonal constrictions encased by paranodal tomacula (excessive myelin folding), a pathological hallmark of HNPP. We hypothesize that the tomacula/axonal constrictions predispose the PMP22 deficient nerves to develop mechanically induced CB. Moreover, we have shown that removal of the p21-activated kinase type-1 (pak1) gene in PMP22 deficient mice eliminates tomacula/axonal constrictions, a novel signaling mechanism. In this proposal we will further investigate the cellular and molecular basis for the development and recovery of CB, the formation of tomacula/axonal constrictions, and the therapeutic potential of PAK inhibitors. Toward these ends, we propose the following specific aims: Aim 1: Test the hypothesis that tomacula/axonal constrictions predispose nerves to mechanically induced CB in PMP22 deficiency. Our preliminary results have shown a hastened mechanically-induced CB and axonal constrictions in tomacula in pmp22 mice. In this aim, we will first determine the relationship between the predisposition of CB and tomacula/axonal constrictions using an additional animal model with tomacula/axonal constrictions and an animal model without these pathologies. We will next investigate potential mechanisms for this predisposition; these include (1) electrophysiological effects caused by axonal deformities in tomacula and (2) possible current leakage out of tomaculous myelin that shunts the depolarizing current to reduce the safety factor for action potential propagation. These mechanisms will be investigated using confocal microscopy and 3-dimentional EM to delineate detail geometric features of axonal deformities in tomacula. Physiological consequences of these tomacula/axon deformities will be evaluated by threshold tracking technique. These results will provide insights into the mechanisms underlying the propensity to mechanically-induce CB in PMP22 deficiency. Aim 2: Test the hypothesis that PAK1 is required for the formation of tomaculum/axonal constriction. PAK1, as a serine-threonine kinase and a member of the PAK family (from PAK1 to 6), interacts with small GTPases for its activation, such as cdc42 and rac. Deficiency of PAK1 in the pak1-/- mice causes no phenotype. After crossbreeding pak1-/- with pmp22 mice, however, double-knockout of both genes eliminates tomacula/axonal constrictions in pmp22 mice. In this aim, we will test whether removal of tomacula will reverse the susceptibility to mechanically-induced CB in PMP22 deficient mice, and further explore this novel signaling pathway. We will attempt to translate this exciting finding to therapy by testing whether newly synthesized PAK inhibitor can reverse tomacula/axonal constrictions in PMP22 deficiency. Aim 3: Identify the mechanisms by which haploinsufficiency of pmp22 delays the recovery of CB. Our experimental results have shown a delayed recovery of mechanically-induced CB in the pmp22 mice. In this aim, cellular and molecular mechanisms that underlie the delayed recovery of CB will be investigated in pmp22 mice. Taken together, these three aims will define cellular and molecular factors that predispose pmp22 nerves to mechanically induced CB, and establish molecular signaling pathway for the formation of tomaculum/axonal constriction in the PMP22 deficiency. Results are expected to deepen our understanding on the molecular basis of CB, which may render insights into the pathogenesis for many demyelinating diseases.